We have examined the respective roles in EAU of the Th1 and the Th17 effector pathways in uveitis by using monoclonal antibodies and mutant mice deficient in various components of these pathways. We found that , depending on the model, autoimmunity to retina could be either Th17 or Th1 driven and that each of these T cell responses was sufficient to mediate disease by itself. This year we have gone on to examine what happens when both types of effector responses are deficient. For this purpose, we generated double-deficient mice, lacking both IL-17 and IFN-&#947;, the hallmark cytokines for Th17 and Th1 responses, We find that these mice still develop full-blown EAU with a mostly granulocytic ocular inflammatory infiltrate. They appear to be producing undiminished amounts of IL-22, a cytokine usually associated with a Th17 response, and enhanced amounts of Th2 type cytokines, including IL-5, IL-13 and TNF-&#945;. We are currently examining which cytokine(s) are driving pathology in this model, by using in vivo neutralization studies. These results shed light on the clinical heterogeneity of human uveitis and may impact on therapy. The innate immune response impacts on adaptive immunity as well as directly affects immunopathogenic processes. We continued our studies in which we found that mouse as well as human NKT cells make a very rapid IL-17 response upon ligation of the T cell receptor (TCR) and/or the IL-23 Receptor. We have now identified a novel population of innate T cells, which are distinct from NKT cells. These cells share some conventional T cell memory markers but are CD4-CD8-, rapidly produce high amounts of IL-17 upon T cell receptor and IL-23 receptor ligation, and do not produce IFN-&#947;. We are currently characterizing this cell population with the goal of defining its role in host defense and tissue pathology. In collaboration with investigators at Case Western Reserve University, Cleveland, OH, we have examined the role of complement in EAU. Mice deficient in the decay-accelerating factor of complement (DAF), i.e., having a higher then normal complement activity, developed significantly more severe EAU and expressed higher levels of inflammatory mediators. Conversely, wild type mice that received soluble DAF protein treatments were protected from disease and exhibited reduced Th1/Th17 responses. It is conceivable that manipulation of DAF levels could constitute an approach to therapy of uveitis An, 2009 #1. Vitamin D3 (VitD3, calcitriol) has been shown to have immunomodulatory properties. We examined the ability of VitD3 to modulate EAU. VitD3 prevented as well as partly reversed disease and suppressed immunological responses. Experiments both in vivo and in vitro demonstrated that the mechanism involved inhibition of the Th17 effector response. This occurred at several levels, including the ability of DC to support priming of Th17 cells, the ability of CD4(+) T cells to commit to the Th17 lineage, and the ability of committed Th17 cells to produce IL-17 Tang, 2009 #2. We continue to examine the effect of retinal glial M&#252;ller cells on uveitogenic T lymphocytes. We have previously shown that retinal glial M&#252;ller cells inhibit activation and proliferation of T cells in an Ag-independent fashion by a contact dependent mechanism. We examined what molecules in M&#252;ller cells might be involved in inhibition, and identified thrombospondin-1 and TGF-&#946;as two possible candidates. We also examined the outcome of this interaction on the suppressed T cells. T cells that have interacted with Muller cells downregulate expression of IL-2 receptor. They do not seem to become anergic, as after separation from the M&#252;ller cells and re-culture they are still able to proliferate. These same cells, when irradiated, are able to inhibit proliferation of fresh T cells, however, this function does not seem to require contact with M&#252;ller celllls, as T cells cultured in the same way, only without M&#252;ller cells, are similarly inhibitory. (Cortes/Rigden et al., manuscript in preparation). The internal microenvironment within the eye is immunoinhibitory, a property that is conferred by on TGF-&#946;as well as other substances within the eye, and is believed to protect they eye from inflammation. We examined the role of retinoic acid (RA), which, as a molecule participating in the chemistry of vision, is abundant in the eye. RA has recently been shown to be important in regulatory phenomena elsewhere in the body, but has not been studied in the eye. Our data revealed that aqueous humor (AH) inhibited activation and acquisition of effector function (IFN-&#947;and IL-17 production) by lymphocytes, which were instead converted to FoxP3+, functionally active T-regulatory cells (Tregs). The process was dependent on RA and on TGF-&#946;and involved, at least in part, TGF-&#946;-mediated upregulation of RA receptor (RAR). However, in contrast to T cells undergoing initial activation in presence of AH, previously activated cells exposed to AH on 2nd stimulation were resistant to effects of AH. Our data support the notion that the role of RA in the eye is not limited to the visual process but also contributes to ocular immune privilege. Nevertheless, differentiated effector cells are relatively insensitive to AH, helping to explain occurrence of uveitis in the face of an inhibitory ocular microenvironment. Immunological responses to S-Ag have been implicated in human uveitis, however, direct study of uveitogenic epitopes in humans is not possible. We have established a "humanized" EAU model in HLA transgenic (Tg) mice. Using bioinformatic methods for epitope prediction we are studying recognition and pathogenicity of S-Ag epitopes, and identifying their core sequences. We have identified permissive HLA-DR3(0301), HLA-DR4(0402), HLA-DQ8 and non-permissive HLA-DR4(0401), HLA-DR2 (*1501, *1502, and *1503) alleles of the HLA-DR and DQ genes, and characterized some of the allele-specific uveitogenic epitopes of human S-Ag. Importantly, the sequences of these epitopes overlap with peptides M and N of S-Ag, and the S-Ag crossreactive B27PD peptide derived from HLA-B27 molecule, that were reported to be recognized by lymphocytes of uveitis patients. We have now developed MHC tetramers loaded with these peptides to study S-Ag specific lymphocytes and have demonstrated that they can detect antigen-specific cells in HLA Tg mice with EAU. We will use these tetramers to attempt detection of antigen-specific cells in uveitis patients of the appropriate HLA types, with the goal of developing this method into a diagnostic tool and biomarker for antigen-specific responses in patients (Mattapallil et al., manuscript in preparation). We have developed T cell receptor (TCR) Tg mice carrying a TCR specific to the major epitope of IRBP (IRBP TCR Tg). These mice express 20-25% of IRBP-specific cells, as detected by a specific peptide-MHC class II-Ig dimer developed in our lab. They develop spontaneous EAU disease by 2 months of age, that can be very severe. These mice represent a new model of spontaneous EAU and are serving as a tool to study the development, migration and function of antigen-specific cells in uveitis. (Horai et al, manuscript in preparation)